The long term objective of this application is to develop methods that enable the rapid identification of the mechanistic type and frequency of antineoplastic drug resistant cells within populations of blast cells obtained from patients with acute nonlymphocytic leukemia (ANLL). The importance of such methodologies cannot be overstated, since they will not only aid in the immediate selection of therapy, but will also give direction to future research in the design of new therapeutic approaches, aimed at overcoming those mechanisms of drug resistance identified. These studies will focus on the two most effective agents currently available for treatment of ANLL, daunorubicin (DNR) and 1-B-D-arabinofuranosylcytosine (ara-C). We will utilize state-of-the-art flow cytometric and cell sorting technology to enhance detection of drug resistant cells.
Our specific aims propose: 1) to perfect methods for detecting DNR resistance by studying factors influencing the intracellular biochemistry or pharmacology of DNR. These studies will focus on detection of markers of multidrug resistance such as P-glycoprotein and intracellular DNR concentration, as well as other biochemical parameters which may serve as markers for other forms of DNR resistance; 2) to perfect methods for detecting ara-C resistance by studying biochemical events related to ara-C metabolism and mechanism of action. To this end, we will attempt to produce a monoclonal antibody that recognizes ara-C incorporated into DNA. Nascent DNA containing ara-C, obtained by the pH-step alkaline elution method, will be used as antigen. We will also utilize the pH-step alkaline elution method to study the effects of ara-C on DNA synthesis; 3) to detect resistance to ara-C or DNR on the basis of their cytotoxicity to leukemic cells. These studies will utilize a sensitive flow cytometric viability assay we have developed that quantifies leukemic cell survival following exposure to cytotoxic drug.
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